Analysis of genetic variation is important in many different applications of molecular biology and medicine, such as mapping nucleic acids, investigating genetic diseases and genetically influenced traits, and assessing the genetic attributes of populations. Assessing this variation depends on the ability to detect polymorphisms, the existence of two or more different alleles of a nucleic acid. Different alleles can be identified according to differences in nucleic acid sequences, and genetic variations occurring in more than 1% of a population (which is the commonly accepted rate of random genetic mutation) are useful polymorphisms for certain applications.
Single nucleotide polymorphisms (SNPs) are an abundant form of nucleic acid sequence variation, occurring at a rate of approximately one per 500 nucleotides in coding sequences, and more abundantly in noncoding sequence. See, e.g., Wang D. G., et al., Science 280:1077-82 (1998). As many as a million SNPs may exist in the human genome.
SNPs of moderate or high abundance (having rare allele frequencies of greater than 10%) are amenable to genotyping using different methodologies, such as DNA arrays (see, e.g., Pastinen, T., et al., Genome Res. 10:1031-42 (2000)), mass spectrometry (see, e.g., Jackson, P. E., et al., Mol. Med. Today 6:271-76 (2000)), and PCR end-plate read methods (see, e.g., Livak, K. J., Genet. Anal. 14:143-49 (1999)). For example, sequencing ten chromosomes (from five persons) will detect approximately two-thirds of all SNPs that have a rare allele frequency of at least 10%. However, SNPs that alter nucleic acid expression or affect structure of the nucleic acid product often have rare allele frequencies of less than 10%. For example, sequencing 10 chromosomes using these known methods will detect less than 10% of SNPs having rare allele frequencies about 1% (which is a frequently used threshold for defining polymorphism). Thus, detecting these rare SNPs with greater efficiency, accuracy, and sensitivity than is now possible would provide a benefit in the investigation of conditions and characteristics related to genetic polymorphisms.
Several methods for SNP detection (such as denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography) are based on the thermodynamic properties of DNA duplexes or single stranded DNA (such as single-strand conformational polymorphism analysis). However, these methods for SNP detection indirectly assess differential melting of heteroduplex DNA, rather than directly detecting such differential melting.